Expression of classical NF‐κB pathway effectors in human ovarian carcinoma

Darb‐Esfahani S, Sinn B V, Weichert W, Budczies J, Lehmann A, Noske A, Buckendahl A‐C, Müller B M, Sehouli J, Koensgen D, Györffy B, Dietel M & Denkert C
(2010) Histopathology 56. 727–739
Expression of classical NF‐κB pathway effectors in human ovarian carcinoma Aims: Functional studies have demonstrated that nuclear factor (NF)‐κB promotes tumour progression in ovarian cancer cells. However, surprisingly little is known of the expression of effectors of the NF‐κB pathway in human ovarian cancer in vivo. Methods and results: Immunohistochemistry and in situ hybridization revealed that in a cohort of 85 primary ovarian carcinomas, total p65 expression was inversely correlated to nuclear and cytoplasmic phospho‐IκBα (P = 0.002 and P = 0.05, respectively), and IκBα mRNA expression (P = 0.032). In contrast, phospho‐p65 expression was paralleled by the expression of nuclear (P = 0.027) and cytoplasmic phospho‐IκBα (P = 0.01). Total p65 expression was an adverse prognostic factor for overall survival (P = 0.018). In contrast, total IκBα and phosphorylated nuclear and cytoplasmic IκBα expression were favourable prognostic markers (P = 0.001, P = 0.031, P = 0.001, respectively). Cytoplasmic phospho‐IκBα expression remained a significant prognostic factor on multivariate analysis (P = 0.010). In cultured, stimulated OVCAR‐3 ovarian cancer cells the cytoplasmic retranslocation of p65 was delayed by inhibition of the nuclear membrane transporter chromosomal region maintenance/exportin1 protein (CRM1). A positive association of p65 and CRM1 expression was demonstrated in ovarian cancer tissue (P < 0.0001). Conclusions: Total and phosphorylated IκBα protein expression might serve as markers for NF‐κB activation in human ovarian carcinoma. Cytoplasmic localization of p65 may be related to deregulated nucleocytoplasmic transport in carcinomas overexpressing CRM1.     

The B‐cell‐activating factor signalling pathway is associated with Helicobacter pylori independence in gastric mucosa‐associated lymphoid tissue lymphoma without t(11;18)(q21;q21)

We previously reported that activation of the B‐cell‐activating factor (BAFF) pathway upregulates nuclear factor‐κB (NF‐κB) and induces BCL3 and BCL10 nuclear translocation in Helicobacter pylori (HP)‐independent gastric diffuse large B‐cell lymphoma (DLBCL) tumours with evidence of mucosa‐associated lymphoid tissue (MALT). However, the significance of BAFF expression in HP independence of gastric low‐grade MALT lymphomas without t(11;18)(q21;q21) remains unexplored. Sixty‐four patients who underwent successful HP eradication for localized HP‐positive gastric MALT lymphomas without t(11;18)(q21;q21) were studied. BAFF expression was significantly higher in the HP‐independent group than in the HP‐dependent group [22/26 (84.6%) versus 8/38 (21.1%); p < 0.001]. Similarly, BAFF receptor (BAFF‐R) expression (p = 0.004) and nuclear BCL3 (p = 0.004), BCL10 (p < 0.001), NF‐κB (p65) (p = 0.001) and NF‐κB (p52) (p = 0.005) expression were closely correlated with the HP independence of these tumours. Moreover, BAFF overexpression was significantly associated with BAFF‐R expression and nuclear BCL3, BCL10, NF‐κB (p65) and NF‐κB (p52) expression. These findings were further validated in an independent cohort, including 40 HP‐dependent cases and 18 HP‐independent cases of gastric MALT lymphoma without t(11;18)(q21;q21). The biological significance of BAFF signalling in t(11;18)(q21;q21)‐negative lymphoma cells was further studied in two types of lymphoma B cell: OCI‐Ly3 [non‐germinal centre B‐cell origin DLBCL without t(11;18)(q21;q21) cell line] and MA‐1 [t(14;18)(q32;q21)/IGH‐MALT1‐positive DLBCL cell line]. In both cell lines, we found that BAFF activated the canonical NF‐κB and AKT pathways, and induced the formation of BCL10–BCL3 complexes, which translocated to the nucleus. BCL10 and BCL3 nuclear translocation and NF‐κB (p65) transactivation were inhibited by either LY294002 or by silencing BCL3 or BCL10 with small interfering RNA. BAFF also activated non‐canonical NF‐κB pathways (p52) through tumour necrosis factor receptor‐associated factor 3 degradation, NF‐κB‐inducing kinase accumulation, inhibitor of κB kinase (IKK) α/β phosphorylation and NF‐κB p100 processing in both cell lines. Our data indicate that the autocrine BAFF signal transduction pathway contributes to HP independence in gastric MALT lymphomas without the t(11;18)(q21;q21) translocation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activation of the NF-kappaB signalling pathway in diffuse large B-cell lymphoma: clinical implications

Aim: To characterize the activation of the nuclear factor (NF)‐κB pathway in diffuse large B‐cell lymphoma (DLBCL) by immunohistochemistry. Methods and results: Sixty‐six DLBCLs treated with anthracycline‐containing chemotherapy were evaluated with antibodies against phosphorylated p65 (P‐p65), p65, p50, p52, IKKα, and phosphorylated IκB (P‐IκB). NF‐κB activation was based on the expression of P‐p65, P‐IκB, and nuclear expression of p65 or p52 in the tumour cells. P‐p65 and P‐IκB were expressed in 13 (20%) and 17 cases (26%), respectively. p65, p52 and IKKα were found in the cytoplasm. A correlation was found between expression of P‐p65 and P‐IκB (P < 0.0001), but not between the two subtypes of DLBCL [germinal centre B cell and non‐germinal centre (GC)]. P‐p65+ tumours showed a better response to chemotherapy (P = 0.025) and a trend to increased event‐free survival (P = 0.08). However, P‐IκB expression was not associated with either clinical response or survival. Bcl‐2 was not preferentially expressed on DLBCL tumours with NF‐κB activation, as determined by expression of P‐p65 and P‐IκB proteins. Conclusions: NF‐κB activation in DLBCL is preferentially mediated through the classical pathway and a novel mechanism involving phosphorylation of p65. Activation of NF‐κB by P‐p65 is associated with good prognosis. NF‐κB activation is not confined to non‐GC DLBCL exclusively.     

NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Suppression of oxLDL‐Induced MMP‐9 and EMMPRIN Expression by Berberine via Inhibition of NF‐κB Activation in Human THP‐1 Macrophages

Upregulation of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) by macrophages leads to atherosclerotic plaque rupture by degradation of the extracellular matrix. NF‐κB activation regulates many key inflammatory genes linked to atherosclerosis. In the present study, the function of berberine, a natural extract from Rhizoma coptidis, on MMP‐9 and EMMPRIN expression, the role of NF‐κB activation in oxLDL‐stimulated macrophages, and the possible mechanism in which NF‐κB activation is involved were investigated. Berberine inhibited the expression of MMP‐9 and EMMPRIN at both mRNA and protein levels. The phosphorylation of IκB‐α and nuclear translocation of p65 protein were reduced by berberine, suggesting that NF‐κB activation was inhibited by berberine in oxLDL‐stimulated macrophages. Overall, berberine suppressed the expression of MMP‐9 and EMMPRIN by at least reducing partly the activity of NF‐κB in oxLDL‐induced macrophages. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

New evidence of connections between increased O‐GlcNAcylation and inflammasome in the oral mucosa of patients with oral lichen planus

Oral lichen planus (OLP) is considered a chronic inflammatory immune‐mediated disease of the oral mucosa. Immunopathogenesis of OLP is thought to be associated with cell‐mediated immune dysregulation. O‐GlcNAcylation is a form of reversible glycosylation. It has been demonstrated that O‐GlcNAcylation promoted nuclear factor kappa B (NF‐κB) signalling. Activation of NF‐кB can induce expression of nucleotide‐binding domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which is a large intracellular multi‐protein complex involving an immune response. Dysregulated expression of the NLRP3 inflammasome was reported to be associated with autoinflammatory diseases. No integrative studies between O‐GlcNAcylation and NLRP3 inflammasome in OLP patients have been reported. The present study aimed to determine the immunohistochemical expression of O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome in oral mucosae of OLP patients. Oral tissue samples were collected from 30 OLP patients and 30 healthy individuals. Immunohistochemical staining and analyses of immunostaining scores were performed to evaluate expression of O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome. According to observations in this study, significantly higher levels of O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome were demonstrated in OLP patients compared with control subjects (P < 0·001). Positive correlations among O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome were also observed in OLP samples (P < 0·01). In conclusion, the present study provides supportive evidence that increased O‐GlcNAcylation is associated with increased expression of NLRP3 inflammasome via the NF‐κB signalling pathway. These findings provide a new perspective on immunopathogenesis of OLP in relation to autoinflammation.     

Transcriptional regulation of pro‐apoptotic Par‐4 by NF‐κB/p65 and its function in controlling cell kinetics during early events in endometrial tumourigenesis

Prostatic apoptosis response‐4 (Par‐4) was first identified in prostatic cancer cells that were induced to undergo apoptosis. Recently, Par‐4 has been suggested to be a tumour suppressor gene that plays a role in the development of endometrial carcinomas (ECs), but the exact mechanism remains to be clarified. Here we examined gene activation signalling cascades and influence on cell kinetics during endometrial tumourigenesis. In normal endometrium, constitutively high levels of Par‐4 expression were observed in epithelial cells through the menstrual cycle, in contrast to the transient up‐regulation in stromal components in the menstrual stage, correlated positively with the phospho‐p65 (pp65) status and apoptosis. In contrast, most ECs exhibited significant down‐regulation as compared to normal endometrium, with positive links only to pp65 expression. In EC cell lines, transfection of the NF‐κB subunit p65 led to transactivation of Par‐4 through specific binding to its promoter region, in contrast to the suppression by active Akt, suggesting that the balance between the two signals may be important to determine Par‐4 expression levels. In addition, transient overexpression of Par‐4 resulted in the induction of not only apoptosis but also senescence, through changes in the expression of bcl‐2 and p21 , respectively. Together, these findings suggest that a signalling cascade involving sequential activation of NF‐κB/p65 and Par‐4 may participate in relatively early events of endometrial tumourigenesis, leading to modulation of cell kinetics including apoptosis and cell cycle progression. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The cyclin‐dependent kinase inhibitor R‐roscovitine down‐regulates Mcl‐1 to override pro‐inflammatory signalling and drive neutrophil apoptosis

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non‐inflammatory phagocytosis by professional phagocytes. Recently, cyclin‐dependent kinase (CDK) inhibitors (e.g. R‐roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF‐κB and MAPK pathways and key endogenous survival proteins (typified by Mcl‐1) are involved in the regulation of neutrophil apoptosis and, in cancer‐cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R‐roscovitine over‐rides TNF‐α and LPS‐induced survival (determined by morphological examination and binding of fluorescently labelled annexin‐V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF‐κB activation (assessed by cytoplasmic IκBα proteolysis and NF‐κB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down‐regulation (at protein and mRNA level) of the survival protein Mcl‐1 but not the pro‐apoptotic bcl‐2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.     

Olfactomedin 1 negatively regulates NF‐κB signalling and suppresses the growth and metastasis of colorectal cancer cells

Uncontrolled growth and distant metastasis are hallmarks of colorectal cancer (CRC), but the mechanisms are poorly understood. Olfactomedin 1 (OLFM1), a member of the olfactomedin domain‐containing protein family, plays an important role in the development of neurogenic tissues. Recently, OLFM1 deregulation was frequently observed in several cancers, and it was induced in colon cell lines after treatment with the demethylating agent 5‐aza‐2'‐deoxycytidine. However, the function of OLFM1 in CRC remains unknown. In this study, we reanalysed published microarray data and found that OLFM1 was significantly down‐regulated in primary CRC samples compared to adjacent non‐cancerous tissues. The results of immunohistochemistry indicated that decreased OLFM1 expression was significantly associated with lymph node status (p = 0.023), distant metastasis (p < 0.001), and AJCC/TNM stage (p = 0.013), and CRC patients with low OLFM1 expression had consistently poor overall survival (OS; p < 0.001) and progression‐free survival (PFS; p < 0.001). Further analysis demonstrated that OLFM1 was epigenetically silenced in CRC tissues and cell lines via promoter hypermethylation. Overexpression and knockdown of OLFM1 attenuated and increased, respectively, CRC cells' proliferation, migration, and invasion in vitro and metastasis to the lung and liver in vivo. Mechanistically, the promotion of growth and metastasis of CRC cells by silencing of OLFM1 was associated with the activation of the non‐canonical NF‐κB signalling pathway. OLFM1 interacted with NF‐κB‐inducing kinase (NIK; MAP3K14) and repressed the phosphorylation of its downstream substrate Ikappa B kinase alpha (IKKα). OLFM1 expression was negatively correlated with the phosphorylation level of IKKα in CRC tissue samples. Knockdown of NIK impaired the ability of OLFM1 to repress NF‐κB signalling, cell growth or migration. Thus, OLFM1 may be a valuable biomarker and therapeutic target for CRC patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Golgi phosphoprotein 3 (GOLPH3) promotes hepatocellular carcinoma cell aggressiveness by activating the NF‐κB pathway

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, in which the NF‐κB pathway plays an important role and is constitutively activated. Better understanding of the molecular pathogenesis of HCC and the NF‐κB pathway are needed to improve patient outcomes. Herein, we identified an unappreciated protein involved in NF‐κB‐induced activation, Golgi phosphoprotein 3 (GOLPH3). The mRNA and protein expression levels of GOLPH3 were frequently up‐regulated in HCC and GOLPH3 expression correlated closely with clinical stage and survival in both the testing and validation cohorts. Ectopic over‐expression of GOLPH3 in PLC/PRF/5 (PLC) and Huh7 HCC cells protected against cisplatin‐induced apoptosis, promoted angiogenesis and proliferation and increased the aggressiveness of HCC cells in vitro and in vivo, whereas inhibition of GOLPH3 led to decreased aggressiveness. Through analysis of two published HCC patient profiles, GOLPH3 expression significantly correlated with NF‐κB signalling. Furthermore, we demonstrated that GOLPH3 promoted K63‐linked polyubiquitination of tumour necrosis factor receptor‐associated factor 2 (TRAF2), receptor interacting protein (RIP) and NF‐κB essential modulator (NEMO) and substantially sustained the activation of NF‐κB in HCC cells. Taken together, our findings provided evidence that GOLPH3 is a prognostic and/or potential therapeutic biomarker for HCC patients and plays an important role in activation of the NF‐κB pathway during HCC progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

HHQ and PQS,two Pseudomonas aeruginosa quorum‐sensing molecules,down‐regulate the innate immune responses through the nuclear factor‐κB pathway

To explore whether bacterial secreted 4‐hydroxy‐2‐alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild‐type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor‐α and interleukin‐6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild‐type did not. The addition of 4‐hydroxy‐2‐heptylquinoline (HHQ) or 2‐heptyl‐3,4‐dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild‐type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor‐κB (NF‐κB) binding to its binding sites and the expression of NF‐κB target genes, and PQS delayed inhibitor of κB degradation, indicating that the effect of HHQ and PQS was mediated through the NF‐κB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.     

PKC,ERK/p38 MAP kinases and NF‐κB targeted signalling play a role in the expression and release of IL‐1β and CXCL8 in Porphyromonas gingivalis‐infected THP1 cells

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.